Solid-phase oligonucleotide syntheses initially employed the use of phosphate triesters (the "triester method") or phosphites (the "phosphite method"). With the discovery of relatively stable mononucleoside phosphoramidite coupling units (see, for example, Beaucage and Caruthers, Tetra. Lett., 1981, Vol. 22, 1859-1862), solid-phase oligonucleotide synthesis became practical and common. Typical solid-phase oligonucleotide synthesis involves reiteratively performing four steps: deprotection, coupling, capping, and oxidation.
Standard methods involve stepwise synthesis of the oligonucleotide in the 5'-direction. In the first step ("deprotection"), the growing oligonucleotide, which is attached at the 3'-end via a 3'-O-group to a solid support, is 5'-deprotected to provide a reactive group (i.e., a 5'-OH group). For example, the 5'-OH group is often protected by reaction with 4,4'-dimethoxytrityl chloride (DMT--Cl) in pyridine, to yield a 5'-ODMT group, which is stable under basic conditions, but which is easily deprotected under acid conditions, for example, in the presence of dichloroacetic acid (DCA) or trichloroacetic acid (TCA).
In the second step ("coupling"), the 5'-deprotected supported oligonucleotide is reacted with the desired nucleotide monomer, which itself has first been converted to a 5'-protected, 3'-phosphoramidite. For example, the 5'-OH group may be protected in the form of a 5'-ODMT group and the 3'-OH group may converted to a 3'-phosphoramidite, such as --OP(OR')NR.sub.2, where R is the isopropyl group, --CH(CH.sub.3).sub.2, and R' is, for example, --H (yielding a phosphoramidite diester), or --CH.sub.3, --CH.sub.2 CH.sub.3, or the beta-cyanoethyl group, --CH.sub.2 CH.sub.2 CN (yielding a phosphoramidite triester). The 3'-phosphoramidite group of the monomer reacts with the deprotected 5'-OH group of growing oligonucleotide to yield the phosphite linkage 5'-OP(OR')O-3'. See, for example, Caruthers, M. H. and S. L. Beaucage, U.S. Pat. No. 4,415,732, issued Nov. 15, 1995.
Not all of the growing oligonucleotides will couple with the provided monomer; those which have not "grown" would yield incomplete oligonucleotides and therefore must be withdrawn from further synthesis. This is achieved by the third step ("capping"), in which all remaining --OH groups (i.e., unreacted 5'-OH groups) are capped, for example, in the form of acetates (5'-OC(O)CH.sub.3,) by reaction with acetic anhydride (CH.sub.3 C(O)--O--C(O)CH.sub.3).
Finally, in the oxidation step, the newly formed phosphite group (i.e., 5'-OP(OR')O-3') of the growing oligonucleotide is converted to a phosphate group (i.e., 5'-OP(.dbd.O)(OR')O-3'), for example, by reaction with aqueous iodine and pyridine.
The four-step process may then be reiterated, since the oligonucleotide obtained after oxidation remains 5'-protected (e.g., 5'-ODMT) and is ready for use in the first deprotection step described above.
When the desired oligonucleotide has been obtained, it may be cleaved from the solid support, for example, by treatment with alkali and heat. This step may also serve to convert phosphate triesters (i.e., when R' is not --H) to the phosphate diesters (--OP(.dbd.O).sub.2 O--), as well as deprotect base-labile protected amino groups of the nucleotide bases.
In the preparation of longer oligonucleotides, the earlier triester method offered better yields owing the availability of appropriate triester dimer and trimer coupling units (see, for example, Hirose et al., Tetra. Lett., 1978, pp. 2449-2452). However, recent developments in oligonucleotide synthesis have provided for the use of nucleotide multimers (i.e., short-chain oligonucleotides), such as nucleotide dimer phosphoramidites, as opposed to only nucleotide monomers, so as to reduce the number of required reiterations and permit both an increase in overall yield and a reduction in chemical manipulation. See, for example, Kumar and Poonian, J. Org. Chem., 1984, Vol. 49, pp. 4905-4912. For example, to obtain the oligonucleotide (dGdT).sub.10 dG, one might start with a dG-type solid-phase support (e.g., dG-CPG) and perform 10 reiterations using dGdT-dimer units (e.g., 5'-protected-dG-dT-3'-phosphoramidite), as opposed to starting with a dG-type solid-phase support and performing 20 reiterations using dG- and dT-monomer units (e.g., 5'-protected-dG-3'-phosphoramidite and 5'-protected-dT-3'-phosphoramidite).
For example, with a coupling efficiency of 99% (assumed equal for monomer and dimer couplings), twenty monomer couplings yields only (0.99).sup.20 or 81.8% yield, whereas ten dimer couplings yields (0.99).sup.10 or 90.4% yield. For a 97% coupling efficiency, the monomer and dimer yields are (0.97).sup.20 =54.4% and (0.97).sup.10 =73.7%, respectively. The ratio of the monomer to dimer yields reflects the "break even yield" required for economical synthesis of the dimer. For example, with a 97% coupling efficiency, the break even yield is 54.4/73.7=73.8%; that is, the benefits of dimer-based synthesis may be more fully realized when one is able to prepare the dimer coupling units from monomers units with 73.8% yield or greater, all other factors being equal.
For example, consider theoretically that for 20 monomer couplings, one might perform 20 steps, each step employing 1000 monomer equivalents; for the corresponding 10 dimer couplings, one would perform 10 steps, each step employing 1000 dimer equivalents, which must themselves be prepared from monomers with some inherent losses. If dimers may be synthesized from monomers with only 80% yield, then the required 10,000 useful dimer equivalents may be prepared from 25,000 monomer equivalents, as compared to the 20,000 monomer equivalents needed for monomer coupling. For a coupling efficiency of 97%, one obtains 544 correct oligonucleotides per 1000 solid-phase support sites via the 20-cycle monomer coupling method which employs 20,000 monomer equivalents (i.e., 2.72.times.10.sup.-5 oligos/site/monomer), whereas one obtains 737 correct oligonucleotides per 1000 sites via the 10-cycle dimer coupling method which employs 25,000 monomer equivalents (i.e., 2.95.times.10.sup.-5 oligos/site/monomer).
The success of these "compact" syntheses has relied on the availability of suitable dimer-phosphoramidites. For example, all sixteen di(deoxynucleotide) dimers (in the form of N-blocked-5'-ODMT-3'-2-chlorophenyl!-2'-deoxynucleotidyl-3'.fwdarw.5'!-N -blocked-2-chlorophenyl!-2'-deoxynucleoside-3'-2-cyanoethyl!phosphates) are commercially available. Typically, these dimers are prepared using a solid-phase oligonucleotide synthesis method. Consequently, use of many of the resulting dimers is relatively expensive as compared to the use of the individual monomers.
The present invention provides improved methods for solution-phase synthesis of short-chain oligonucleotides, such as nucleotide dimers, which are useful as coupling units in solution-phase or solid-phase oligonucleotide synthesis.